One clinical hallmark of patients with severe pneumonia or sepsis-induced lung injury is a robust acute inflammatory host response triggered by the invading pathogens. Here, the innate immunity system is activated to secrete large amounts of pro-inflammatory cytokines (i.e. a cytokine storm) after invading pathogens activate receptors on immune effector cells (T-cells, macrophages, etc.). The TRAF family of adaptors molecules are pivotal and highly conserved proteins that link external surface signals (e.g. via Toll receptors) to cytokine release via NF-kB dependent signaling. Thus, maneuvers designed to selectively modulate the availability of TRAF family proteins might lessen the severity of sepsis. However, to date, very little is known regarding the molecular regulation of the TRAF family at the level of protein stability. In the process of studying bacterial sepsis, we discovere that an orphan ubiquitin E3 ligase subunit, termed FBXO3, is activated and sufficient to ubiquitinate and mediate proteasomal degradation of another relatively new ubiquitin E3 ligase subunit, termed FBXL2. Further, we discovered that FBXL2 is anti-inflammatory as it targets the TRAF family of proteins for their disposal in epithelia and monocytes. Thus, our preliminary work demonstrates that P. aeruginosa, via activation of FBXO3, results in FBXL2 ubiquitination and degradation resulting in increased immunoreactive TRAFs, increased cytokine production, and impaired lung stability. These data led to our novel hypothesis that P. aeruginosa induced cytokine release involves FBXO3 mediated ubiquitination and degradation of FBXL2, which in turn increases levels of TRAF proteins which are pro-inflammatory. We will determine if an orphan ubiquitin E3 ligase subunit, FBXO3, coordinates with GSK3b to trigger ubiquitination dependent degradation of FBXL2, which in turn activates TRAF proteins to stimulate cytokine release (Aim 1). We will also determine if mutation or inhibition of FBXO3 will attenuate bacterial-induced acute lung injury (Aim 2). This proposal provides a new model of innate immunity as it relates to cytokine signaling. Execution of these studies will lay the foundation fo a significant mechanistic advance regarding the molecular regulation of the innate immune response during severe bacterial infection. Results from these studies are intended to serve as the basis for strategies directed at the development of highly selective novel small molecule inhibitors of FBXO3 to lessen the severity of bacterial or sepsis-induced acute lung injury.